Real time detection of antigens

ABSTRACT

Antigens can be captured and detected from complex samples, such as food and environmental samples, in about 30 minutes using apparatus and methods that include flow of the samples through a module containing antibodies coupled to beads. The samples flow through the modified beads at about 0.2 to 1.2 liters/minute, which fluidizes the bead bed. The antigens are captured by the antibodies, and then detection of the captured antibodies is carried out with chemiluminescence, fluorescence, or spectrophotometric techniques.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. Ser. No.10/163,253, filed Jun. 4, 2002, abandoned, which is acontinuation-in-part of U.S. Ser. No. 09/292,172, filed Apr. 15, 1999,now U.S. Pat. No. 6,399,317, which claims the benefit of U.S.Provisional Application No. 60/081,889, filed Apr. 15, 1998, all ofwhich are hereby incorporated by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not applicable.

BACKGROUND OF THE INVENTION

[0003] This invention relates to detection of antigens. Moreparticularly, the invention relates to compositions and methods fordetection of selected antigens in real time. In an illustrativeembodiment, the invention relates to compositions and processes forsensitive detection of microbes and contaminants in complex samples,such as food samples, environmental samples, and the like, within about30 minutes.

[0004] Bacterial spores are the most heat-stable form of microorganisms,are ubiquitous in the environment, and are therefore of great concern infood products, such as milk, that receive extensive heat treatments toprolong shelf life. Spore counts in milk from around the world vary fromzero to greater than 22,000 cfu/ml depending on the climate of theregion. S. A. Chen et al., A Rapid, Sensitive and Automated Method forDetection of Salmonella Species in Foods using AG-9600 AmpliSensorAnalyzer, 83 J. Appl. Microbiol. 314-321 (1997). Bacillusstearothermophilus spores are one of the most heat-resistant bacterialspores and are found in high numbers in soil and water. Contaminating B.stearothermophilus spores survive extreme heat to germinate and grow atelevated product storage temperatures, which occur in foods transportedin equatorial regions of the world.

[0005] While B. stearothermophilus is not commonly a problem, otherbacilli often lead to food-borne illness or spoilage in a variety offoods. Bacillus cereus, Bacillus licheniformis, Bacillus subtilis, andBacillus pumilus have all been implicated in outbreaks of food-borneillness and are commonly isolated from raw and heat treated milk. M. W.Griffiths, Foodborne Illness Caused by Bacillus spp. other than B.cereus and Their Importance to the Dairy Industry, 302 Int. Dairy Fed.Bulletin 3-6 (1995). B. cereus is also responsible for a sweet curdlingdefect in milk as well as being pathogenic. W. W. Overcast &K. Atmaram,The Role of Bacillus cereus in Sweet Curdling of Fluid Milk, 37 J. MilkFood Technol. 233-236 (1973). A mesophilic heat resistant bacillussimilar to Bacillus badius, has been isolated from extreme temperatureprocessed milk (D₁₄₇=5 sec; P. Hammer et al., Pathogenicity Testing ofUnknown Mesophilic Heat Resistant Bacilli from UHT-milk, 302 Int. DairyFed. Bulletin 56-57 (1995)). B. badius is a mesophilic organism andgrows readily at room temperature, making it a likely candidate forspoiling temperature-processed foods. There have been 52 confirmed casesof B. badius in UHT milk in Europe and two cases outside of Europe. P.Hammer et al., supra. Lack of a rapid spore assay that can be used inmilk contributes to the difficulty of prediction of post processingspoilage, thereby limiting the shelf life and product safety. H. Hofstraet al., Microbes in Food-processing Technology, 15 FEMS Microbiol. Rev.175-183 (1994). Such an assay could be used in a hazard analysiscritical control point (HACCP) plan allowing raw materials with highspore loads to be diverted to products that do not pose a food safetyrisk to consumers.

[0006] The standard method for quantifying spores in milk involvesheat-shocking and an overnight plate count. G. H. Richardson, StandardMethods for the Examination of Dairy Products (1985). This istime-consuming and yields historical information. The food industryneeds microbiological assays to yield predictive information for maximumbenefit in HACCP analysis and risk assessment. An enzyme-linkedimmunosorbent assay (ELISA) capable of detecting greater than 10⁶ cfu/mlof B. cereus spores in foods has been reported, but was unacceptable dueto antibody cross-reactivity. Y. H. Chang & P. M. Foegeding,Biotin-avidin Enzyme-linked Immunosorbent Assay for Bacillus Spores inBuffer and Food, 2 J. Rapid Methods and Autom. Microbiol. 219-227(1993).

[0007] Techniques to increase sensitivity of immunosorbent assays havefocused on more efficient reporter labels, such as faster catalyzingreporter-enzymes; signal amplification, such as avidin- orstreptavidin-biotin enzyme complexes; and better detectors, such asluminescence and fluorescence. L. J. Kricka, Selected Strategies forImproving Sensitivity and Reliability of Immunoassays, 40 Clin. Chem.347-357 (1994); P. Patel, Rapid Analysis Techniques in Food Microbiology(1994). Immunomagnetic antigen capture is used extensively to separateand identify Escherichia coli and Salmonella from foods. C. Blackburn etal., Separation and Detection of Salmonellae Using ImmunomagneticParticles, 5 Biofouling 143-156 (1991); P. M. Fratamico et al., RapidIsolation of Escherichia coli O157:H7 from Enrichment Cultures of FoodsUsing an Immunomagnetic Separation Method, 9 Food Microbiol. 105-113(1992); L. Krusell & N. Skovgaard, Evaluation of a New Semi-automatedScreening Method for the Detection of Salmonella in Foods within 24 h,20 Inter. J. Food Microbiol. 124-130 (1993); A. Lund et al., RapidIsolation of K88⁺ Escherichia coli by Using Immunomagnetic Particles, 26J. Clin. Microbiol. 2572-2575 (1988); L. P. Mansfeild & S. J. Forsythe,Immunomagnetic Separation as an Alternative to Enrichment Broths forSalmonella Detection, 16 Letters Appl. Microbiol. 122-125 (1993); A. J.G. Okrend et al., Isolation of Escherichia coli O157:H7 Using O157Specific Antibody Coated Magnetic Beads, 55 J. Food Prot. 214-217(1992); E. Skjerve & Olsvic, Immunomagnetic Separation of Salmonellafrom Foods, 14 Inter. J. Food Microbiol. 11-18 (1991); D. J. Wright etal., Immunomagnetic Separation as a Sensitive Method for IsolatingEscherichia coli O157 from Food Samples, 113 Epidemiol. Infect. 31-39(1994). However, these methods involve either a preincubation or asubsequent incubation step (usually 18 to 24 hours) to increase the cellnumbers for detection. Immunomagnetic capture greatly shortens E. coliand Salmonella testing, but long incubation times limit this method forpredictive information. Immunocapture has also been used to quantitateBacillus anthracis spores in soil samples using luminescent detection,J. G. Bruno & H. Yu, Immunomagnetic-electrochemiluminescent Detection ofBacillus anthracis Spores in Soil Matrices, 62 App. Environ. Microbiol.3474-3476 (1996), but these efforts have led to tests that have adetection limit of 10³ cfu/ml.

[0008] Considerable progress in the development of biosensors formicrobial detection has been achieved in the last decade. Thesebiosensors can be applied to medical, process control, and environmentalfields. They must possess ideal features such as high specificity,simplicity, sensitivity, reliability, reproducibility, and speed. S. Y.Rabbany et al., Optical Immunosensors, 22 Crit. Rev. Biomed. Engin.307-346 (1994). With the use of antibodies as the recognition elementfor specific capture, numerous applications have been developed fordetection of pathogenic bacteria. M. R. Blake & B. C. Weimer,Immunomagnetic Detection of Bacillus stearothermophilus Spores in Foodand Environmental Samples, 63 J. Appl. Environ. Microbiol. 1643-1646(1997); A. Burkowski, Rapid Detection of Bacterial Surface ProteinsUsing an Enzyme-linked Immunosorbent Assay System, 34 J. Biochem.Biophys. Methods 69-71 (1997); S. A. Chen et al., A Rapid, Sensitive andAutomated Method for Detection of Salmonella Species in Foods UsingAG-9600 AmpliSensor Analyzer, 83 J. Appl. Microbiol. 314-321 (1997); L.A. Metherell et al., Rapid, Sensitive, Microbial Detection by GeneAmplification using Restriction Endonuclease Target Sequence, 11 Mol.Cell Probes 297-308 (1997); F. Roth et al., A New MultiantigenImmunoassay for the Quantification of IgG Antibodies to CapsularPolysaccharides of Streptococcus pneumoniae, 176 J. Inf. Dis. 526-529(1997).

[0009] Methods for continuous flow immunoassay for rapid and sensitivedetection of small molecules have been developed. For example, A. W.Kusterbeck et al., 135 J. Immunol. Methods 191-197 (1990), describessuch a method in which detection of the antigen occurred within a matterof minutes. The assay is based on the binding of labeled antigen to animmobilized antibody, with subsequent displacement of the labeledantigen when antigen is present in the sample flow. Signal detectionoccurs downstream of the antigen recognition event.

[0010] In standard displacement flow immunoassays, the analyte of up to1000 molecular weight in the sample creates an active dissociation oflabeled antigens from an antigen binding site of an immobilizedantibody, after which the labeled substance is measured downstream. W.A. Kaptein et al., On-line Flow Displacement Immunoassay for FattyAcid-binding Protein, 217 J. Immunol. Methods 103-111 (1998), describesdisplacement in a flow system for detection of a small protein,cytoplasmic heart-type fatty acid-binding protein (15,000 molecularweight), a plasma marker for myocardial injury. This displacement systemuses an inverse set-up: enzyme-labeled monoclonal antibodies areassociated to immobilized antigen and are displaced by analyte in thesample.

[0011] F. Vianello et al., Continuous Flow Immunosensor for AtrazineDetection, 13 Biosens. Bioelectron. 45-53 (1998), describes detection ofthe hapten, atrazine, under continuous flow conditions using amicro-column containing immobilized monoclonal antibodies againstatrazine and atrazine labeled with alkaline phosphatase. The equilibriumof the antibody-hapten system was achieved by continuous flow of thetracer (alkaline phosphatase-labeled atrazine) through the micro-columncontaining the immobilized antibodies. The activity of the tracer wasmonitored continuously downstream of the micro-column with anamperometric detector using p-hydroquinone phosphate as substrate. Whenpulses of unlabeled atrazine were added to the tracer flowingcontinuously through the micro-column, tracer bound to the antibody wasdisplaced, with a consequent change in the detector signal.

[0012] C. H. Pollema & J. Ruzicka, Flow Injection Renewable SurfaceImmunoassay: A New Approach to Immunoanalysis with FluorescenceDetection, 66 Anal. Chem. 1825-1831 (1994), describes automaticheterogeneous immunoassays using a flow injection technique on arenewable surface. This assay relies on a minute amount of beads to forma reactive surface, which is interrogated by fluorescence spectrometry.Following the assay, the spent reactive surface is fluidically removedand replaced with a new layer of beads.

[0013] B. Mattiasson & M. P. Nandakumar, Binding Assays in HeterogeneousMedia Using a Flow Injection System with an Expanded Micro-bedAdsorption Column, 8 Bioseparation 237-245 (1999), describes acompetitive binding assay in a flow injection system wherein theadsorption step was carried out in an expanded bed column to increasethe versatility of the assay an enable it to deal with samplescontaining particulate matter.

[0014] In view of the foregoing, it will be appreciated thatcompositions and methods for real time detection of selected antigens,such as contaminants in food and the environment, would be a significantadvancement in the art.

BRIEF SUMMARY OF THE INVENTION

[0015] The present invention comprises compositions and methods forcapture and detection of antigens from complex liquid samples within amatter of minutes and without the need for culturing of organisms.

[0016] An illustrative method according to the present invention forcapturing and concentrating a selected antigen from an aqueous mediumcontaining a mixture of antigens comprises:

[0017] (a) causing a first volume of the aqueous medium containing themixture of antigens to flow through a module containing at least twoantibody-bead conjugates, wherein each of the antibody-bead conjugatescomprises a bead, a polymeric spacer covalently coupled to the bead, andan antibody covalently coupled to the polymeric spacer, wherein theantibody is configured for binding the selected antigen, at a first flowrate such that the antibody-bead conjugates form a fluidized bed and theselected antigen binds to the antibody-bead conjugates;

[0018] (b) washing the antibody-bead conjugates having the selectedantigen bound thereto by causing a wash medium to flow through themodule at a second flow rate such that the antibody-bead conjugateshaving the selected antigen bound thereto form a fluidized bed; and

[0019] (c) holding the washed antibody-bead conjugates having theselected antigen bound thereto in a second volume of a second washmedium, wherein the second volume is smaller than the first volume.

[0020] Another illustrative method according to the present inventionfor detecting a selected antigen in aqueous medium containing a mixtureof antigens comprises:

[0021] (a) causing the aqueous medium containing the mixture of antigensto flow through a module containing at least two antibody-beadconjugates, wherein each of the antibody-bead conjugates comprises abead, a polymeric spacer covalently coupled to the bead, and an antibodycovalently coupled to the polymeric spacer, wherein the antibody isconfigured for binding the selected antigen, at a first flow rate suchthat the antibody-bead conjugates form a fluidized bed and the selectedantigen binds to the antibody-bead conjugates;

[0022] (b) washing the antibody-bead conjugates having the selectedantigen bound thereto by causing a first wash medium to flow through themodule at a second flow rate such that the antibody-bead conjugateshaving the selected antigen bound thereto form a fluidized bed; and

[0023] (c) detecting the selected antigen bound to the antibody-beadconjugates by enzyme-linked immunosorbent assay.

[0024] An illustrative apparatus according to the present invention foruse in capturing and detecting antigens comprises:

[0025] (a) a housing comprising a wall defining an interior chamber andcomprising an inlet opening for conducting a liquid medium into theinterior chamber and an outlet opening for conducting the liquid mediumout of the interior chamber, wherein at least a portion of the wall isoptically transparent;

[0026] (b) at least two antibody-bead conjugates disposed in thehousing, each comprising a bead, a polymeric spacer covalently coupledto the bead, and an antibody coupled to the polymeric spacer;

[0027] (c) a liquid circulation circuit coupled to the housing forconducting the liquid medium into the interior chamber through the inletopening and for conducting the liquid medium out of the interior chamberthrough the outlet opening at a selected flow rate; and

[0028] (d) a photomultiplier tube mounted adjacent to the opticallytransparent portion of the wall for measuring photons produced in theinterior chamber.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0029]FIG. 1A illustrates specific capture of B. stearothermophilusspores from a mixed population containing equal amounts of B.stearothermophilus and B. subtilis spores, where B. subtilis sporecounts in PBST are represented by the dotted bar, B. stearothermophilusspore counts in PBST are represented by the open bar, B. subtilis sporecounts in milk are represented by the hatched bar, and B.stearothermophilus spore counts in milk are represented by the solidbar. FIG. 1B shows the concentration of spores in the wash: (□) B.subtilis in PBST, (⋄) B. stearothermophilus in PBST, (∘) B. subtilis inmilk, and (Δ) B. stearothermophilus in milk.

[0030]FIG. 2 shows fluorescence detection of captured B.stearothermophilus spores in skim milk by a biotin-avidin amplifiedsandwich ELISA using 3×10⁶ (□) and 1.4×10⁷ (∘) immunomagnetic beads(IMBs); data points represent the mean of two replications, and errorbars represent standard error of the means.

[0031]FIG. 3 shows fluorescence detection of captured B.stearothermophilus spores from various food and environmental samplesusing 3×10⁶ IMBs: (⋄) muck clay, R²=0.82; (∘) pepper, R²=0.96; (□) skimmilk, R²=0.99; (Δ) whole milk; (▪) acidic sandy soil (pH 3.7); datapoints represent the mean of two replications, and error bars representstandard error of the means.

[0032]FIG. 4 shows a schematic representation of an illustrative modulefor use in detecting antigens according to the present invention.

[0033]FIG. 5 shows immunoflow (2 L/min) detection of B. globigii sporesin 0.1 M phosphate buffer (pH 7.2, R²=0.9; slope=0.03).

[0034]FIG. 6 shows a bovine serum albumin (BSA) standard curve at therange of 1.5-100 ng/μl; each point is the average absorbance (450 nm) intriplicate, and PBST (0.2% Tween 20) was the blocking reagent to blockthe nonspecific binding sites.

[0035]FIG. 7 shows a BSA standard curve at the range of 0.075-2.5 ng/μl;each point is the average absorbance (450 nm) in triplicate; PBST (0.2%Tween 20) was the blocking reagent.

[0036]FIG. 8 shows a schematic diagram of an illustrative compositionfor use in binding antigens to a solid support according to the presentinvention.

[0037]FIG. 9 shows a schematic diagram of detection of antigens capturedwith the composition of FIG. 8.

[0038]FIGS. 10 and 11 show a module or bead container for holding thebead-bound antibodies for capturing and detecting antigens according tothe present invention. FIG. 10 shows the beads settled by gravity at thebottom of the module, whereas FIG. 11 shows the positions of beads in afluidized condition when liquid is passing through the module.

[0039]FIG. 12 shows a schematic diagram of an illustrative apparatus foruse in detecting antigens according to the present invention.

[0040]FIG. 13. shows capture of E. coli O157:H7 from buffer on thesurface of bead-bound antibodies.

[0041]FIG. 14 shows the average capture efficiency E. coli O157:H7 frombuffer, beer, and apple juice using bead-bound antibodies.

[0042]FIG. 15 shows signal detection from the surface of beads.

[0043]FIG. 16 shows determination of cell density by the method of thepresent invention.

[0044] FIGS. 17A-C show calibration plots of the relative captureactivity versus concentration of antigen. Two types of glass beads,succinylated () and PEG-coupled (▪), were used. FIG. 17A shows resultsfor ovalbumin (OVA). FIG. 17B shows results for B. globigii spores. FIG.17C shows results for E. coli O157:H7.

[0045]FIG. 18 shows a standard curve for BSA capture withtosyl-activated, polythreonine-modified immunomagnetic beads. Standarderror of the mean at each data point is masked by the symbol.

[0046]FIG. 19 shows a standard curve of OVA using 3 mm PEG-modifiedglass beads. Error bars represent standard error of the mean.

[0047]FIGS. 20A&B show standard curves of B. globigii spores in PBST(FIG. 20A) and E. coli O157:H7 in meat extract (□) and PBST (▪) (FIG.20B) using 3 mm PEG-modified glass beads. Standard error of the mean foreach data point is masked by the symbol.

[0048]FIG. 21 shows standard curves of B. globigii spores inenvironmental and industrial water samples using PEG-modified glassbeads: PBST (▪), river water (♦), tank water (∘), slush tank (Δ). Errorbars represent standard error of the mean.

DETAILED DESCRIPTION

[0049] Before the present compositions and methods for real timedetection of antigens are disclosed and described, it is to beunderstood that this invention is not limited to the particularconfigurations, process steps, and materials disclosed herein as suchconfigurations, process steps, and materials may vary somewhat. It isalso to be understood that the terminology employed herein is used forthe purpose of describing particular embodiments only and is notintended to be limiting since the scope of the present invention will belimited only by the appended claims and equivalents thereof.

[0050] The publications and other reference materials referred to hereinto describe the background of the invention and to provide additionaldetail regarding its practice are hereby incorporated by reference. Thereferences discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention.

[0051] It must be noted that, as used in this specification and theappended claims, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. For example,reference to an apparatus containing “a bead” includes reference to twoor more of such beads, reference to “a spacer” includes reference to oneor more of such spacers, and reference to “an antibody” includesreference to two or more of such antibodies.

[0052] In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outherein.

[0053] As used herein, “antibody” means an immunoglobulin molecule thatinteracts and binds only with the antigen that induced its synthesis inlymphoid tissue and/or with antigens closely related to it. Includedwithin this definition of antibody all antibody types, e.g., IgG, IgA,IgM, etc.; IgG subclasses, e.g., IgG1, IgG2, etc.; F(ab) fragments;F(ab′)₂ fragments; F(ab′) fragments; light chain dimers; single chainantibodies, and the like. This definition also includes antibodies thatreact with low and high affinity with an antigen. Further, suchantibodies can be polyclonal or monoclonal.

[0054] As used herein, “cfu” means colony forming units.

[0055] As used herein, “PBS” means phosphate buffered saline: 0.01 MNa₂HPO₄, 0.15 M NaCl, pH 7.2.

[0056] As used herein, “PBST” means phosphate buffered saline containing0.1% Tween 20.

[0057] As used herein, “BSA” means bovine serum albumin.

[0058] As used herein, “ELISA” means enzyme-linked immunosorbent assay.

[0059] As used herein, “comprising,” “including,” “containing,”“characterized by,” and grammatical equivalents thereof are inclusive oropen-ended terms that do not exclude additional, unrecited elements ormethod steps. “Comprising” is to be interpreted as including the morerestrictive terms “consisting of” and “consisting essentially of.”

[0060] As used herein, “consisting of” and grammatical equivalentsthereof exclude any element, step, or ingredient not specified in theclaim.

[0061] As used herein, “consisting essentially of” and grammaticalequivalents thereof limit the scope of a claim to the specifiedmaterials or steps and those that do not materially affect the basic andnovel characteristic or characteristics of the claimed invention.

[0062] A fluidized or extended volume reactor filled with beads thathave been modified with antibodies was developed for capturing antigens,such as specific microorganisms and biological molecules. The beads canbe glass, ceramic, and the like, and are relatively large compared tothose used in many antibody capture processes, typically in the range ofabout 1-7 millimeters (mm) in diameter. The antibodies are attached tothe beads through a spacer. Typical spacers include polymers such asdextran, polyethylene glycol (PEG), and polyamino acids, such aspolyserine and polythreonine.

[0063] Flow speed is typically about 0.2 to about 1.2 liters per minute.More typically, the flow speed is about 0.3 to about 0.7 liters perminute. The flow of the sample and wash solutions through thebead-containing module can be generated by use of vacuum pump,peristaltic pump, and other similar methods known in the art.

[0064] Detection of captured antigens can be by methods well known inthe art, such as by surface ELISA using chemiluminescence (photometric),fluorescence, and spectrophotometric detection.

[0065] Luminescence is related to fluorescence in that both producephotons or light. In the case of fluorescence, however, energy must alsobe applied to excite the photons to escape the molecular structure. Thisis most often done with a laser and specific wavelengths of light.Luminescence does not require light input since chemicals or biologicalmolecules provide the energy to excite the molecule. Unlike detectionsystems based on fluorescence, chemiluminescence methods do not requireexternal light sources for excitation energy. The signals are generatedinternally as light-producing chemical reactions occur.

[0066] Detection of antigens according to the present inventiontypically involves a luminescence reaction, although fluorescence orcolorimetric detection can also be used. The reporter enzyme used in thereaction determines which chemiluminescent substrate is employed.Horseradish peroxidase (HRP) and alkaline phosphatase (AP) are the twomost common reporter enzymes. A common substrate is luminol (cyclicdiacyl hydrazide), which is oxidized during the enzyme reaction. Thisoxidation converts the luminol substrate into an excited intermediatedianion. As the intermediate returns to its ground state, it emits lightat a maximum of 425 nm. Another substrate is Lumigen APS-5 (Lumigen,Inc., Southfield, Mich.), which emits light at a maximum of 430 nm.Chemiluminescence is typically about 2 orders of magnitude moresensitive than fluorescence and more than 4 orders of magnitude moresensitive than chromogenic reactions. This sensitivity allows for lowerdetection limits in standard assays, such as ELISA.

EXAMPLE 1

[0067] Bacteria.

[0068] The bacteria used in the experiments described herein aredescribed in Table 1. Commercial preparations of spores of B.stearothermophilus ATCC 10149, B. cereus ATCC 11778, and B. subtilis6633 were purchased from Fisher Scientific, Pittsburgh, Pa. Viable sporenumbers and germination estimates were obtained by plating on platecount agar (PCA) overnight at 65° C. and 30° C., respectively. All otherspores except for B. globigii spores (Table 1) were prepared byspread-plating a single colony isolate on PCA and incubating the coveredplate at 30° C. for approximately 2 weeks. Spores were swabbed from thesurface of the agar and washed repeatedly in distilled water to removewater-soluble components. Spores were pelleted and separated from celldebris by centrifugation (1,500×g for 20 min at 4° C.; D. E. Gombas & R.F. Gomez, Sensitization of Clostridium perfringens Spores to Heat byGamma Radiation, 36 Appl. Environ. Microbiol. 403-407 (1987)). Presenceof spores was confirmed by heating to 80° C. for 15 min and then platingon PCA (G. H. Richardson, supra). Presence of an exosporium on the sporewas tested by phase contrast microscopy with crystal violet staining (C.Du & K. Nickerson, Bacillus thuringiensis HD-73 Spores HaveSurface-localized Cry Ac Toxin: Physiological and PathogenicConsequences, 62 Appl. Environ. Microbiol. 3722-3726 (1996)). Sporetiters were estimated by plating spores on plate count agar (PCA) andincubating overnight at 37° C. Based on these experiments, it wasestimated that 10¹¹ spores have a mass of 1 g. TABLE 1 Inc. temp.Species (° C.) Source Exosporium B. stearothermophilus 65 ATCC 10149^(a)− B. cereus 30 ATCC 11778^(a) + B. subtilis 30 ATCC 6633^(b) + B.circulans 30 ATCC 4513^(b) − B. coagulans 30 ATCC 7050^(b) − B. globigii30 Dugway^(c) B. licheniformis 30 OSU^(d) − B. mascerans 30 OSU^(d) + B.polymyxa 30 ATCC 842^(b) + B. pumilus 30 OSU^(d) −

EXAMPLE 2

[0069] Polyclonal Antibodies Production.

[0070] Polyclonal antibodies against B. cereus spores, B. subtilisspores, and B. stearothermophilus spores were made at the Utah StateBiotechnology Center (Logan, Utah). BALB/c mice were injected in theintraperitoneal cavity with 1×10⁷ cfu/ml cells or spores in sterilesaline (0.5 ml) three times at 3-week intervals. E. Harlow & D. Lane,Antibodies, A Laboratory Manual (1988). Total ascites IgG was purifiedusing a protein A/G column (Pierce Chemical, Rockford, Ill.). Antibodieswere desalted and concentrated to 1 mg/ml in 0.1 M NaPO₄, pH 7.0 in a 30kD Centricon filter (Amicon, Beverly, Mass.) at 4,500×g at 4° C.

[0071] Goat antibodies to Bacillus globigii spores were obtained fromDugway Proving Grounds (Tooele, Utah).

EXAMPLE 3

[0072] Monoclonal Antibody Production.

[0073] Monoclonal antibodies were produced against B. stearothermophilusby suspending the cells or spores in PBS to an optical density of 0.93at 550 nm before intraperitoneally injecting female BALB/c mice with0.250 mg (whole cell wet weight) without adjuvant. The mice wereimmunized 3 times at 3-week intervals. Seven days after the lastimmunization they were test bled, and the serum was titered by ELISA 3days before fusion. Booster injections were administered byintraperitoneal injection with 0.1 mg cells in PBS. Fusion with acompatible murine myeloma cell line (P3X63-Ag8.653) was done in thepresence of polyethylene glycol. Selection for hybrid cells was done inHAT medium. G. Kohler & C. Milstein, Continuous Cultures of Fused CellsSecreting Antibody of Pre-defined Specificity, 256 Nature 495-97 (1975)(hereby incorporated by reference). Positive colonies were determined byELISA and were subcloned twice before freezing in liquid nitrogen.

EXAMPLE 4

[0074] Antibody Specificity.

[0075] Antibody specificity was tested by measuring the cross reactivityagainst Bacillus spores listed in Table 1 using a standard ELISA. Asuspension of each spore type (10⁶ cfu/ml), suspended in 50 mM NaCO₃ (pH9.5), was nonspecifically bound to wells of a microtiter plate for 12 hat 4° C. Wells containing spores were blocked with 2% bovine serumalbumin (BSA) in PBS for 4 h at 25° C., and washed four times with PBScontaining 0.1% Tween 20 (PBST). Anti-B. stearothermophilus antibodies(1:10,000 serum dilution in PBS) were added to wells, slowly agitatedfor 2 h at 25° C., and washed four times with PBST.Horseradish-peroxidase-labeled (HRP) anti-whole mouse IgG (SigmaChemical Co., St. Louis, Mo.) was added to label anti-B.stearothermophilus antibodies for 2 h, then the wells were washed fourtimes with PBST. O-Phenylenediamine dihydrochloride (Sigma) colordevelopment was measured using a b* color scale (blue to yellow) at 37°C. for 1 h in an automated reflectance calorimeter (Omnispec 4000Bioactivity monitor; Wescor, Inc., Logan, Utah).

[0076] The anti-B. stearothermophilus antibodies did not cross reactwith any of the spore types tested (Table 1) including common aerobicspores found in raw foods. Lack of cross reactivity may be partly due tothe absence of an exosporium on the B. stearothermophilus spores (Table1). However, antibodies raised against B. subtilis and B. cereus, whichhave exosporia, were also specific for the injected spore types,suggesting that the surface antigens of the exosporia are sufficientlydifferent as to not crossreact.

EXAMPLE 5

[0077] Antibody Attachment to Beads Via Biotin-Streptavidin.

[0078] Anti-B. stearothermophilus antibodies purified from total serumwere biotinylated with NHS-LC-Biotin (Pierce Chemical, Rockford, Ill.).Efficiency of surface biotinylation was determined using the HABA assay(Pierce), except that the 2-mercaptoethanol step was omitted to avoiddenaturing antibodies. This modified procedure gave the number ofsurface biotin moieties per antibody (Sigma Technical Support).

[0079] Biotinylated antibodies were coupled to streptavidin-boundmagnetic beads (Dynabeads Streptavidin™, Lake Success, N.Y.) accordingto the supplier's directions.

EXAMPLE 6

[0080] Antibody Attachment to Bead Via Poly(threonine).

[0081] Sodium meta-periodate (5 mg) was used to oxidize carbohydratemoieties on the anti-B. stearothermophilus antibodies. G. T. Hermansonet al., Immobilized Affinity Ligand Techniques (1992) (herebyincorporated by reference). Sodium meta-periodate was removed afteroxidation by washing five times with 0.1M NaPO₄, pH 7.0, in a 30 kDCentricon filter (4,500×g, 4° C.), and the oxidized antibodies were thenimmediately crosslinked to beads magnetic beads.

[0082] PolyThr (MW(vis) 12,100;Sigma Chemical, St. Louis, Mo.) wascovalently coupled to 2.8-μm, tosyl-activated polystyrene Dynabeads(Dynal, Lake Success, N.Y.) in 50 mM borate buffer (pH 9.5) via theterminal amine as described by the product instructions. Four washes(three times for 10 min, and once for 30 min) with TBS buffer (pH 7.5)were used to block remaining tosyl-active sites. Adenine dihydrazine(ADH; 0.5 M in 0.1 M MES, pH 4.75; Sigma) was linked to the carboxyterminus of the bound PolyThr using an ethylene diamine carbodiimidemediated reaction (G. T. Hermanson et al., supra). Oxidized antibodieswere mixed with the ADH-activated beads at room temperature for 12 h toallow crosslinking between the oxidized carbohydrate moiety of the IgGand the ADH terminus of the PolyThr spacer (G. T. Hermanson et al.,supra). After crosslinking, the modified immunomagnetic beads (IMBs)were stored rotating (50 rpm) in PBST with 0.02% sodium azide at 40° C.until use.

EXAMPLE 7

[0083] Antibody Attachment to Bead Via Poly(serine).

[0084] In this example, the procedure of Example 6 was followed exceptthat poly(serine) was substituted for poly(threonine).

EXAMPLE 8

[0085] Antibody Attachment to Bead Via Dextran.

[0086] Ceramic beads, 7 mm in diameter (Coors Ceramics Corp., Golden,Colo.), were washed in acidic methanol (HCl:methanol, 1:1) for 30 min atroom temperature (RT) to strip the bead surface. The acidic methanol waspoured off and the beads were rinsed several times with filtered water(dH₂O). The beads were further washed with concentrated sulfuric acidthree times for 30 min, rinsed several times with dH₂O, and finallyboiled in dH₂O for 30 min to introduce hydroxyl groups onto the surface.

[0087] For silanization and crosslinking, beads were air dried, washedonce in toluene and incubated in 3% 3-mercapto propyl trimethoxysilane(3% MTS in toluene) for 2 h at RT. Subsequently the beads were preparedfor the addition of the crosslinker γ-maleimidobutyric acid N-hydroxysuccinimide ester (GMBS; Sigma Chemicals, St. Louis, Mo.). Beads werewashed twice in toluene to remove unbound MTS, air dried, and thenincubated for 1 h at RT in 2 mM GMBS (in 100% ethanol). Finally, thebeads were finally washed in 100% ethanol and then in PBS.

[0088] Dextran was used as a spacer between the crosslinker and theantibody. Sodium-m-periodate (Sigma Chemicals, St. Louis, Mo.) was usedto oxidize the carbohydrate moieties on dextran (37.5 kDa, SigmaChemicals, St. Louis, Mo.) for 3 h at RT while shaking. The salt wasremoved by washing four times with dH₂O in 30 kDa Centricon filters(Amicon Inc., Beverly, Mass.) and immediately bound to the crosslinkedbeads. Adipic acid dihydride (ADH, 0.5 M in sodium phosphate, pH7.2;Sigma Chemicals, St. Louis, Mo.) was then added to introduce anamine group to the bead surface, which could then react with theoxidized antibody. All unreacted sites were blocked with 1% Tris/BSA, pH8.5.

EXAMPLE 9

[0089] In this example, anti-B. stearothermophilus antibodies were mixedwith tosyl-activated magnetic beads (Dynal) according to the directionssupplied with the beads such that the amine groups on the antibodiesreacted with tosyl groups on the surface of the beads. The resultingmodified beads contained the antibodies covalently bonded to the surfaceof the beads.

EXAMPLE 10

[0090] In this example, anti-Fc IgG was bound to magnetic beadsaccording to the procedure of Example 9. After unbound IgG was washedoff, the beads were reacted with anti-B. stearothermophilus antibodiessuch that the anti-Fc IgG bound the anti-B. stearothermophilusantibodies. The anti-Fc IgG thus formed a spacer between the magneticbeads and the anti-B. stearothermophilus antibodies.

EXAMPLE 11

[0091] In this example, anti-B. stearothermophilus antibodies attachedto magnetic beads were tested for their ability to capture B.stearothermophilus spores. The antibody/bead conjugates (i.e.,immunomagnetic beads or IMBs) were prepared according to the procedureof Examples 5, 6, 9, and 10. ELISA using HRP-labeled anti-IgG confirmedthe presence of bound antibodies on the surfaces of the beads.

[0092] IMBs (3×10⁶ beads) were added to 1 ml of sample comprising 10⁴ or10⁶ B. stearothermophilus spores in PBST. These mixtures were incubatedfor 30 min at 25° C. with rotation at 50 rpm. The IMBs were removed fromthe sample for 2 min with a magnetic particle concentrator (DynalMPC-E-1) and washed four times with PBST to reduce IMB clumping andblock spore adhesion to tube walls (E. Skjerve et al., Detection ofListeria monocytogenes in Foods by Immunomagnetic Separation, 56 Appl.Environ. Microbiol. 3478-3481 (1990)). After each wash, IMBs weretransferred to a new microfuge tube. The presence of bound spores onIMBs was confirmed in duplicate by plate counts and phase contrastmicroscopy. TABLE 2 Table 2 shows the results of these experiments. AbNo. spores Attachment Modification Ab Orientation bound Biotin- NHS-LC-Non- 0^(a) Streptavidin Biotinylation directional 0^(b) Ab-NH₂ to NoneNon- 0^(a) Tosyl groups directional 0^(b) on beads Anti-Fc IgG NoneDirectional 0^(a) spacer 0^(b) PolyThr-ADH Carbohydrate Directional160^(a ) crosslinker oxidation 3600^(b  )

[0093] These results show that of the conjugates tested only antibodiesbound to beads through a poly(threonine) spacer were able to capturespores. These data suggest that spacer length and flexibility may play arole in the antibody-antigen interaction.

EXAMPLE 12

[0094] In this example, the procedure of Example 11 was followed exceptthat conjugates containing poly(serine) (Example 7) and dextran (Example8) spacers were substituted for the conjugate containing thepoly(threonine) spacer. The results obtained with the poly(serine)—anddextran-containing conjugates were substantially similar to thoseobtained with the poly(threonine)—containing conjugate.

EXAMPLE 13

[0095] In this example, equal numbers of B. subtilis and B.stearothermophilus spores were mixed in PBST and in milk. Immunocaptureusing anti-B. stearothermophilus antibodies conjugated to magnetic beadswas carried out according to the procedure of Example 11 except thatsamples containing milk were given 5 minutes to separate the beads fromthe medium using the magnetic particle concentrator due to the slowerbead recovery. After capture of spores using the immunomagnetic beadconjugate, the conjugates were washed with PBST and the wash supernateswere plated on PCA. This washing procedure was repeated three times suchthat a total of four wash supernates were assayed.

[0096]FIG. 1A shows the conjugate specifically captured B.stearothermophilus spores from PBST and milk containing equal numbers ofB. stearothermophilus and B. subtilis spores. FIG. 1B shows that about99% of non-specifically bound spores were removed from the conjugatewith each wash, leaving B. stearothermophilus spores captured by theconjugate after four washes.

[0097] The anti-B. stearothermophilus antibodies did not cross reactwith any of the spore types tested (Table 1) including common aerobicspores found in raw foods.

EXAMPLE 14

[0098] Product Testing.

[0099] In this example, muck clay, ground pepper, skim milk, whole milk,and acidic sandy soil were tested for detection of bacterial spores.Fluid products were tested with no modification. Powdered products weresuspended at 1 g/ml. Anti-B. stearothermophilus antibody conjugatedbeads prepared according to the procedure of Example 6 were added to 1ml of each product and mixed gently at 25° C. Bound spores werequantitated using calorimetric (as described above) or fluorescencedetection. For fluorescence detection, spores bound to IMBs were labeledwith secondary biotinylated anti-B. stearothermophilus antibodies. TheIMBs were then washed with PBST and resuspended in an ABC-alkalinephosphatase complex solution (Vector Laboratories, Inc., Burlingame,Calif.) for 30 min. The IMBs were washed three times with PBST andresuspended in 100 μl of 0.2 M Tris buffer containing 0.1% BSA (pH 8.5)to remove unbound enzyme complex. A 40-μl suspension of the IMBs wasadded to 3 ml of Fluorophos substrate (Advanced Instruments, Norwood,Mass.) and fluorescence monitored for 2 min at 38° C. in a FluorophosFLM200 fluorometer (Advanced Instruments, Norwood, Mass.).

[0100] As shown in FIG. 2, using immunocapture-sandwich ELISA, spores inUHT skim milk were quantified down to 8×10³ cfu/ml in 2 h with nopre-enrichment steps and no sample preparation. Increasing the number ofbeads in the assay increased the fluorescence activity, suggesting thatthis could further increase the assay sensitivity (P. M. Fratamico etal., supra; E. Skjerve et al., supra).

[0101] The slope of the generated curves was similar for all samplestested, indicating that sample background did not grossly influenceantigen binding (FIG. 3). Therefore, approximate spore loads can beobtained without calibrating the assay to each product. Foods containingfat, such as raw whole milk, required a longer time for separation ofthe IMBs and gentle removal of supernate to avoid trapping the beads inthe fat and removing them with the supernate. Separation of IMBs fromfatty products required 5 min rather than the 2 min used for nonfatsamples. Soil samples containing a high percentage of iron finesinterfered with bead recovery, although other soil types tested did not.These data support the use of this assay to test for B.stearothermophilus spores in food and environmental sample.

[0102] Since the assay has been designed to be used with raw ingredientsthat may vary in temperature, the ability of the IMBs to capture B.stearothermophilus spores at temperatures ranging from 4° C. to 55° C.was tested. IMBs were added to 1 ml UHT skim milk containing 5×10⁴ B.stearothermophilus spores and incubated between 4° C. to 55° C. whilerotating (50 rpm) for 30 min. The IMB were washed four times with PBST,plated on PCA, and incubated overnight at 65° C. B. stearothermophiluscolonies were counted to quantitate bound spores. Regardless of thetemperature of the sample, the number of spores captured from UHT skimmilk containing 5×10⁴ B. stearothermophilus spores did not varysignificantly. This means that sample preparation time can be reduced.These data suggest that this approach is over 100 times more sensitivethan the only other rapid spore assay (Y. H. Chang & P. M. Foegeding,supra), is about 10 times faster than any spore assay with equivalentsensitivity (G. H. Richardson, supra), and can be used to quantitate asingle species of spore in a mixed spore population in chemicallycomplex backgrounds.

[0103] While detection of spores was achieved with immunomagnetic beads,it was believed that sensitivity and efficiency could be improved byusing a fluidized bed capture system. Hence, the capture step wasfluidized by immobilizing antibodies onto larger beads ranging is sizefrom 1-7 mm. Use of a fluidized bed module (FIG. 4) further increasedthe sensitivity of the assay to less than a single cell per ml of liquidand allowed the assay to be done without pre-incubation and to obtain afinished result within 30 min in all the samples tested (FIG. 5). FIG. 4shows a schematic representation of an illustrative module 30 comprisinga housing 34 having an inlet opening 38 for flow of a liquid medium tobe tested into the module. A plate 46 having holes therein to permitflow of the liquid through the module is placed with the plane of theplate generally perpendicular to the direction of flow of the liquid.The beads 50 are placed upstream of the plate. Arrow 54 shows thedirection of flow of liquid through the module. The size of the holes inthe plate is selected to be smaller than the size of the beads such thatthe beads cannot pass through the outflow opening 42. In an illustrativeembodiment of the module, the holes in the plate were 3 mm in diameter,and the beads were 7 mm in diameter. The housing and plate should beconstructed of materials, such as stainless steel and high durabilityplastics, having high durability and compatibility with liquids ofvarious types.

EXAMPLE 15

[0104] In this example, 0.1 M phosphate buffer, pH 7.2, containingvarious concentrations of B. globigii spores was passed through animmunoflow module containing 7 mm ceramic beads having anti-B. globigiiantibodies conjugated thereto according to the procedure of Example 6.The buffer was pumped through the module at 2 L/min. After capture ofthe spores, all of the beads were removed from the module, and a solidphase ELISA using biotinylated anti-B. globigii antibodies to amplifythe signal was performed according to the procedure of Blake & Weimer,supra. The signal was read at 410 nm in a Biospec 1601 (ShimadzuScientific Corp., Columbia, Md.) and compared to a standard curve. FIG.5 shows that spores could easily be detected at concentration of lessthan 1 cfu/ml.

[0105] In a companion experiment, the following foods were tested forthe presence of B. globigii spores by immunoflow capture: raw wholemilk, skim milk, raw hamburger, canned green beans, canned corn, cannedpeas, canned carrots, canned mixed vegetables, canned spinach, beer,fermented sausage, Vienna sausage, raw chicken, canned chicken, cannedpork and beans, canned kidney beans, fresh sliced mushrooms, and cannedtomato sauce. Fluid products were tested without modification. Otherproducts were dissolved or suspended at 1 g/ml. Fifty ml of product waspumped through the module at a rate of 2.5 L/min. Bound spores werequantitated using fluorescence detection as described above. B. globigiispores were detected in each of these foods at a concentration of 1spore/ml.

[0106] This is a significant improvement over prior results and provideda method for increasing the sample size that could be used. Use ofimmunoflow at 2-4 L/min allowed detection in less time and in thepresence of fat or protein that interfered with immunomagnetic detectionand some foods. A characteristic dip at 10³ spores/ml was found, whichis commonly observed. The cause for this deviation is unknown but islinked to the lower flow rate used, since this dip is not noticeable atflow rates>4L/min. The dynamic range is at least nine decades,suggesting this module will not easily be overloaded in the field.

EXAMPLE 16

[0107] In this example, the procedure of Example 15 was carried outexcept that river water (pH 8.5) with added B. globigii spores (10³spores/ml) was pumped through the module at a flow rate from 1 to 4L/min for times ranging from 1 to 180 minutes. Detection and sporecapture increased as the flow rate increased, with the maximum detectionat 15 minutes and a flow rate of 4 L/min. Detection decreased as theflow rate decreased and as the flow time increased. These data suggest acomplex interaction between the capture surface and the spore isoccurring, but that it is not matrix dependent. Similar results wereobserved with detection of B. globigii spores in PBS and penicillin inmilk at 114 L/min. Additionally, the results obtained with penicillindetection suggests that the range of flow rates for capture anddetection is large, at least 1-114 L/min.

EXAMPLE 17

[0108] To demonstrate the use of immunoflow with small protein targets,BSA detection was done using immunoflow with 5 mm glass beads modifiedwith dextran and anti-BSA antibodies and a flow rate of 2 L/min. Theassay detected BSA over a broad range (FIG. 6), and had a lowerdetection limit of at least 0.075 ng/ml without the avidin/biotincomplex for amplification (FIG. 7). These data confirm the use ofimmunoflow detection for use with chemicals (penicillin), small proteins(BSA), and microbes (Bacillus spores) at flow rates varying from 1-114L/min.

[0109]FIG. 8 shows an illustrative embodiment of the present inventionfor capturing and detecting an antigen from a sample. This illustrativecomposition 100 comprises an antibody 104 coupled to the surface of asolid support, for example, a bead 108. The antibody 104 is coupled tothe bead 108 by means of a spacer 112. As described above, spacers caninclude dextran, polythreonine, polyserine, polyethylene glycol,biotin/avidin linkages, and the like. The spacer 112 is coupled to thesurface of the bead 108 by a chemical linker 116, and the antibody 104is also coupled to the spacer 112 by a chemical linker 120. A bacterium124 captured by the composition 100 is shown bound to the antibody 104.

[0110]FIG. 9 shows detection of the bacterium 124 captured asillustrated in FIG. 8. Detection is by a high flow rate solid phaseELISA according to methods well known in the art. A secondary antibody128 is linked to an enzyme 132 by means of a chemical linker 136 forproviding an amplification complex 140 for providing a detectable signalwhen a suitable substrate is placed in contact with the amplificationcomplex 140. Illustrative substrates that can be used in accordance withthe present invention include those that yield visual, calorimetric,luminescent, and fluorescent signals upon digestion of the substrate bythe enzyme.

[0111] Methods for detecting antibody/antigen or immune complexes arewell known in the art. The present invention can be modified by oneskilled in the art to accommodate the various detection methods known inthe art. The particular detection method chosen by one skilled in theart depends on several factors, including the amount of sampleavailable, the type of sample, the stability of the sample, thestability of the antigen, and the affinity between the antibody andantigen.

[0112] While these techniques are well known in the art, examples of afew of the detection methods that could be used to practice the presentinvention are briefly described below.

[0113] There are many types of immunoassays known in the art. Forexample, a common type of immunoassay is a non-competitive sandwich orcapture assay, such as enzyme-linked immunosorbent assays (ELISA). In anon-competitive capture ELISA, unlabeled antigen is captured by anantibody bound to a solid support, such as the surface of the bead asillustrated in FIG. 8. After the immune complexes have formed, excesssample is removed and the bead is washed to remove nonspecifically boundantigen. If the concentration of the antigen in the sample issufficiently dilute, however, it may not be necessary to removenonspecifically bound antigens because such antigens are present in suchlow amounts. The immune complexes are then reacted with an appropriateenzyme-labeled antibody (secondary antibody), which recognizes the sameor a different epitope on the antigen as the primary antibody. Thesecondary antibody reacts with antigens in the immune complexes. After asecond wash step, the enzyme substrate is added. The enzyme linked tothe secondary antibody catalyzes a reaction that converts the substrateinto a product. Hence, enzyme activity is directly proportional to theamount of antigen in the biological sample. D. M. Kemeny & S. J.Challacombe, ELISA and Other Solid Phase Immunoassays (1988).Illustratively, the product is fluorescent or luminescent, which can bemeasured using technology and equipment well known in the art. It isalso possible to use reaction schemes that result in a colored product,which can be measured spectrophotometrically, but such calorimetricreactions are not preferred.

[0114] Typical enzymes that can be linked to secondary antibodiesinclude horseradish peroxidase, glucose oxidase, glucose-6-phosphatedehydrogenase, alkaline phosphatase, β-galactosidase, and urease.Secondary antigen-specific antibodies linked to various enzymes arecommercially available from, for example, Sigma Chemical Co. andAmersham Life Sciences (Arlington Heights, Ill.).

[0115] Fluorescence immunoassays can also be used when practicing themethod of the present invention. Fluorescence immunoassays are similarto ELISAs except the enzyme is substituted for fluorescent compoundscalled fluorophores or fluorochromes. These compounds have the abilityto absorb energy from incident light and emit the energy as light of alonger wavelength and lower energy. Fluorescein and rhodamine, usuallyin the form of isothiocyanates that can be readily coupled to antigensand antibodies, are most commonly used in the art. D. P. Stites et al.,Basic and Clinical Immunology (1994). Fluorescein absorbs light of 490to 495 nm in wavelength and emits light at 520 nm in wavelength.Tetramethylrhodamine absorbs light of 550 nm in wavelength and emitslight of 580 nm in wavelength. Illustrative fluorescence-based detectionmethods include ELF-97 alkaline phosphatase substrate (Molecular ProbesInc., Eugene, Oreg.); PBXL-1 and PBXL-3 (phycobilisomes conjugated tostreptavidin) (Martek Biosciences Corp., Columbia, Md.); FITC and TexasRed labeled goat anti-human IgG (Jackson ImmunoResearch Laboratories,Inc., West Grove, Pa.); and B-Phycoerythrin and R-Phycoerythrinconjugated to streptavidin (Molecular Probes Inc.). ELF-97 is anonfluorescent chemical that is digested by alkaline phosphatase to forma fluorescent molecule. Because of turn over of the alkalinephosphatase, use of the ELF-97 substrate results in signalamplification. Fluorescent molecules attached to secondary antibodies donot exhibit this amplification.

[0116] Phycobiliproteins isolated from algae, porphyrins, andchlorophylls, which all fluoresce at about 600 nm, are also being usedin the art. I. Hemmila, Fluoroimmunoassays and ImmunofluorometricAssays, 31 Clin. Chem. 359 (1985); U.S. Pat. No. 4,542,104.Phycobiliproteins and derivatives thereof are commercially availableunder the names R-phycoerythrin (PE) and Quantum Red™ from, for example,Sigma Chemical Co.

[0117] In addition, Cy-conjugated secondary antibodies and antigens areuseful in immunoassays and are commercially available. Cy-3, forexample, is maximally excited at 554 nm and emits light of between 568and 574 nm. Cy-3 is more hydrophilic than other fluorophores and thushas less of a tendency to bind nonspecifically or aggregate.Cy-conjugated compounds are commercially available from Amersham LifeSciences.

[0118] Illustrative luminescence-based detection methods include CSPDand CDP star alkaline phosphatase substrates (Roche MolecularBiochemicals); and SuperSignal® horseradish peroxidase substrate (PierceChemical Co., Rockford, Ill.).

[0119] Chemiluminescence, electroluminescence, andelectrochemiluminescence (ECL) detection methods are also attractivemeans for quantifying antigens and antibodies in a sample. Luminescentcompounds have the ability to absorb energy, which is released in theform of visible light upon excitation. In chemiluminescence, theexcitation source is a chemical reaction; in electroluminescence theexcitation source is an electric field; and in ECL an electric fieldinduces a luminescent chemical reaction.

[0120] Molecules used with ECL detection methods generally comprise anorganic ligand and a transition metal. The organic ligand forms achelate with one or more transition metal atoms forming anorganometallic complex. Various organometallic and transitionmetal-organic ligand complexes have been used as ECL labels fordetecting and quantifying analytes in biological samples. Due to theirthermal, chemical, and photochemical stability, their intense emissionsand long emission lifetimes, ruthenium, osmium, rhenium, iridium, andrhodium transition metals are favored in the art. The types of organicligands are numerous and include anthracene and polypyridyl moleculesand heterocyclic organic compounds. For example, bipyridyl, bipyrazyl,terpyridyl, and phenanthrolyl, and derivatives thereof, are commonorganic ligands in the art. A common organometallic complex used in theart includes tris-bipyridine ruthenium (II), commercially available fromIGEN, Inc. (Rockville, Md.) and Sigma Chemical Co.

[0121] Advantageously, ECL can be performed under aqueous conditions andunder physiological pH, thus minimizing biological sample handling. J.K. Leland et al., Electrogenerated Chemiluminescence: AnOxidative-Reduction Type ECL Reactions Sequence Using Triprophyl Amine,137 J. Electrochemical Soc. 3127-3131 (1990); WO 90/05296U.S. Pat. No.5,541,113. Moreover, the luminescence of these compounds may be enhancedby the addition of various cofactors, such as amines.

[0122] In practice, a tris-bipyridine ruthenium (II) complex, forexample, may be attached to a secondary antibody using strategies wellknown in the art, including attachment to lysine amino groups, cysteinesulfhydryl groups, and histidine imidazole groups. After washingnonspecific binding complexes, the tris-bipyridine ruthenium (II)complex would be excited by chemical, photochemical, and electrochemicalexcitation means, such as by applying current to the bead. E.g., WO86/02734. The excitation would result in a double oxidation reaction ofthe tris-bipyridine ruthenium (II) complex, resulting in luminescencethat could be detected by, for example, a photomultiplier tube.Instruments for detecting luminescence are well known in the art and arecommercially available, for example, from IGEN, Inc.

[0123]FIGS. 10 and 11 show a module 150 or bead holder into which thebead-bound antibodies are placed. The module comprises a transparentplastic tube body 154 that holds the beads 158 in a column format. Thetube body comprises a top end 162 and a bottom end 166. A top cap 170 isdisposed on the top end of the tube body, and a bottom cap 174 isdisposed on the bottom end of the tube body. The bottom cap 174comprises an inlet 178 for permitting liquid to enter the module, andthe top cap 170 comprises an outlet 182 for permitting liquid to exitthe module. The outlet 182 and the inlet 178 are configured for beingreceived in the lumen of tubing for linking the module to liquidhandling systems, as will be described in more detail momentarily. Aporous screen 186 is disposed in the cavity 190 formed by the tube bodyand the top cap and the bottom cap adjacent to both the top end 162 andthe bottom end 166 of the tube body. These porous screens 186 areconfigured to permit liquid to pass therethrough while retaining thebeads 158 in the cavity 190.

[0124] Once the antibodies have been attached to beads as describedherein, the beads are loaded into the module. Typically, 10 to 280 beadsare used. Illustratively, 55 beads (2 g of 3 mm diameter beads) may beused.

[0125]FIG. 12 shows the module 200, as described above, connected tocomponents of a system for use in detecting an antigen in a sample. Thesample 204 is contained in a vessel 208. A section of tubing 212 iscoupled to the inlet 216 of the module 200, and the distal end 220 ofthe tubing 212 is placed in the sample 204. Another section of tubing224 is coupled to the outlet 228 of the module 200, and the distal end232 of the tubing 224 is coupled to a vacuum pump 236. A photomultipliertube 240 or PMT is mounted adjacent to the module 200 such that when thebeads are at rest, the PMT 240 is above the level of the beads 244. ThePMT 240 is coupled to signal detection electronics 248 for detecting andcounting signals produced by the PMT 240.

[0126] The apparatus of FIG. 12 is used by placing a sample 204 in thevessel 208, and then causing the vacuum pump 236 to draw a vacuumthrough the sections of tubing 224 and 212 and the module 200, therebydrawing the sample liquid 204 through tubing 212, through the module200, and through tubing 224. The sample can be recirculated through themodule through another section of tubing 252 or can be collected in atrap 256. A valve 260 in tubing line 252 controls whether the sample isrecirculated or trapped. The flow of the sample through the beads 244causes the beads to be fluidized in the cavity of the module. A portionof the antigens contained in the sample are bound by bead-boundantibodies in the module. After capture of the antigens, the beads canbe washed by placing the distal end 220 of tubing 212 in another vesselcontaining a wash solution. Operation of the vacuum pump draws the washsolution through the beads for washing away debris. After washing of thebeads, enzyme-labeled secondary antibodies are drawn through the modulein a similar manner. The enzyme-labeled secondary antibodies bind to thecaptured antigens. Another washing step can then be carried out. Next, asolution containing a substrate for the enzyme is passed through themodule such that the substrate comes in contact with the enzyme. Thesubstrate is digested by the enzyme to result in a detectable signal,such as a luminescent signal as described above. At this point, thevacuum pump is turned off such that the beads in the module settle bygravity to the bottom of the module. Once this has occurred, the PMT isactivated for detecting the signal, such as a luminescent signal.Detection of the luminescent signal by the PMT results in the productionof an electronic signal, which is transmitted to the signal detectionelectronics apparatus for detection, counting, and analysis of theelectronic signals. The number of electronic signals detected andcounted by the signal detection electronics is proportional to thenumber of antigens captured in the module.

[0127] Illustratively, the vacuum pump can be set to draw a vacuum of 2to 5 inches of mercury. It has been determined through experience that asetting of 5 inches of mercury is satisfactory for capturing a targetantigen on the surface of beads. This amount of vacuum fluidizes thebead bed, and permits capture of sufficient antigen for detection inabout 5 minutes with 50 ml samples. FIG. 13 shows capture of E. coliO157:H7 from buffer on the bead surface with fluorescently labeledcells. Each flow rate has an optimum cycling time for achieving capture.A flow rate of about 150 ml/min is convenient for use with a 50 mlsample. The data of FIG. 13 were verified by doing plate counts,according to methods well known in the art, in various foods in the sameexperimental design, but looking at the remaining fluid in the tube atthe maximum capture settings for 150 ml/min (FIG. 14). In each foodtype, the fluidized bed of beads with anti-E. coli O157:H7 attachedthereto captured the cells during the sampling time at 150 ml/minute. Ineach sample type, the cells were specifically captured onto the beadsurface. Comparison of FIGS. 13 and 14 indicates that the beads capturethe cells at approximately the same efficiency in both methods.Therefore, the cells were specifically captured by the antibody-modifiedbeads.

[0128] Once the target antigen is captured and the debris is washedaway, the other reagents are added in the same manner, i.e., they arecaused to flow into the module with the vacuum of the same setting. Theantibodies bind the antigens, and any excess is washed away from thebeads.

[0129] Next, the substrate, such as Lumigen APS-5, is added and thenheld in the module as the signal develops due to the amplificationcomplex (FIG. 15). This may be done with many types of molecules, buthorseradish peroxidase and alkaline phosphatase are especiallyconvenient. The signal is generated and is stable within about 3minutes. The useful portions of the curve are shown in the circles (FIG.15). The analysis can be done with a running slope or an endpoint. Theinformation needed to calculate the running slope is available withinthe first 2 seconds of the curve.

[0130] Once this curve is generated, it is used to determine the amountof bacteria captured onto the bead surface (FIG. 16). Comparison of theinitial slopes or the endpoint allow the final result to be determined.Based on these calculations, the cell density in the original sample isdetermined and reported. Use of the initial slopes is morediscriminating, but the endpoint is adequate for rough estimation of themid-range concentrations.

[0131] The signal is detected with a photon-counting PMT specific forluminescence, for example a Hamamatsu model H7360.

[0132] Table 3 shows the results of detection of E. coli O157:H7 by theprocedure of the present invention as compared to the results achievedwith known commercial tests. In all cases, the present invention, termedImmunoFlow, detected the added organism in each food within 30 minuteswithout the need for culturing the organism. The ImmunoFlow tests weredone without pre-enrichment, whereas the immunoprecipitation and lateralflow ELISA and all experiments with bean sprouts were done with 24 hr ofpre-enrichment. TABLE 3 Immunopreci- Lateral Food type ImmunoFlow BAMpitation Flow ELISA Apple juice − − − − control Apple juice + + + + Beer− − − − control Beer + + + + Hamburger − − − − control Hamburger + + + +Bean + + + + sprouts

EXAMPLE 18

[0133] In a 1-liter flask, 94 ml of double-distilled water and 1.88 g ofdextran (Sigma, St. Louis, Mo.) were combined and mixed by swirling theflask until all of the dextran was solubilized. The flask was thenwrapped in aluminum foil and 3.12 g of NaIO₄ was added. The flask wasthen capped with foil and placed on a shaker at low speed for 1 hour.Next, the flask was removed from the shaker and 125 g of APTES glassbeads (3 mm diameter) was added. The flask was then returned to theshaker for 1 hour of agitation at low speed. Following agitation, thebeads were removed from the flask and washed with 1.25 liter ofdouble-distilled water and then 125 ml of 50 mM sodium phosphate, pH7.2. After washing, the beads were returned to the original flask, whichwas rinsed prior to the beads being replaced, and then 125 ml of 4 mMADH, 50 mM sodium phosphate (pH 7.2). The pH of the buffer was thenchecked to ensure that the pH was about neutral, and then the flask wasreturned to the shaker for low speed agitation for 2 hours.

[0134] While the beads were shaking, the primary antibody solution wasprepared. The primary antibody (0-2 mg), 5 mg of NaIO₄, and enough PBSto raise the volume to 1.0 ml were combined in a 1.5 ml plastic tube(Eppendorf). The contents were mixed by vortexing until all theingredients had dissolved. Next, a 10-ml desalting column was preparedby washing with 50 ml of PBS (pH 7.2). The washed column was then loadedwith the 1.0 ml solution of primary antibody, followed by elution with 8ml of PBS (pH 7.2). The eluate was collected in a reclosable 15-mlplastic tube, and the presence of the antibody in the eluate wasconfirmed by checking the absorbance at 280 nm.

[0135] The flask containing the beads was then removed from the shaker,and 125 mg of NaBH₄ was added to the flask. The flask was then returnedto the shaker for 30 minutes at low speed. The beads were then removedfrom the flask and washed successively with 1.25 liter ofdouble-distilled water, 250 ml of 50 mM sodium phosphate (pH 7.2)containing 1 M NaCl, and 250 ml PBS (pH 7.2). The washed beads were thenplaced in a fresh 1-liter flask, to which was added 113 ml of PBS (pH7.2) and the primary antibody solution. The flask was then placed on ashaker and the contents were agitated at low speed for 1 hour. Thefoil-capped flask was then chilled overnight at 4° C.

[0136] The next day, 125 mg of NaBH₄ was added to the flask, and theflask was placed on a shaker and agitated at low speed for 30 minutes.

[0137] Following agitation, the beads were washed successively with 1.25liter of 50 mM sodium phosphate (pH 7.2), 250 ml of 50 mM sodiumphosphate (pH 7.2) containing 1 M NaCl, and 250 mL Tris-HCl (pH 7.2).After washing, the beads were poured into a flat glass tray and coveredwith filter-sterilized 2% BSA/0.02% NaN₃. The beads were then separatedinto aliquots in sterile plastic containers and covered with excess 2%BSA/0.02% NaN₃. The containers were then shaken at low speed for 1 hourwith swirling every 30 minutes. The beads were then stored at 4° C.until use.

EXAMPLE 19

[0138] Materials and Methods

[0139] Chemical Reagents

[0140] Borosilicate glass beads (3 mm diameter, 7.5×10⁻⁴ m²/g) wereobtained from VWR Scientific Products. 3-Aminopropyl-triethoxysilane(APTES), succinic anhydride, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), morpholineethanesulfonic acid (MES) ovalbumin,(OVA), and bovine serum albumin (BSA) were obtained from Sigma ChemicalCo. (St. Louis, Mo.). PEG-dicarboxymethyl (MW 3,400) was obtained fromShearwater Polymers, Inc. (Huntsville, Ala.) and the BCA protein assaykit was obtained from Pierce Chemical, Co. (Rockford, Ill.). Bacillusglobigii (BG) spores were provided by Dugway Proving Ground (Dugway,Utah). E.coli 0157:H7 was purchased from ATCC, and rehydrated in TrypticSoy Broth (TSB) at 37° C. All other reagents required in the couplingand wash buffers were analytical grade.

[0141] Antibodies

[0142] Monoclonal mouse anti-chicken egg ovalbumin (anti-OVA, cloneOVA-14) and monoclonal mouse anti-BSA (anti-BSA, clone BSA-33) werepurchased from Sigma Chemical Co (St. Louis, Mo.). Polyclonal goatanti-E.coil 0157:H7 was obtained from Kirkegaard & Perry Laboratory(Gaithersburg, M.) Monoclonal goat anti-Bacillus globigii was kindlyprovided by Dugway Proving Grounds (Dugway, Utah).

[0143] Immobilization

[0144] Glass beads, 200 g, were cleaned in concentrated nitric acid for1 hr in a boiling water bath. Beads were derivatized with3-aminopropyltriethoxy silane according to M. K. Walsh & H. E.Swaisgood, Characterization of a chemically conjugatedbeta-galactosidase bioreactor, 17 J. Food Biochem. 283-292 (1993). Halfof the beads, 100 g, were succinylated with succinic anhydride in 0.1 Msodium acetate buffer, pH 4.0, for 2 hours. Dry succinic anhydride, 10g, was added to 150 ml of sodium acetate buffer for succinylation. TheAPTES and succinylated glass beads were dried overnight at 80° C. andstored at room temperature.

[0145] Dicarboxymethyl-PEG was covalently attached to APTES-modifiedglass beads using a one-step EDC reaction according to G. T. Hermansonet al., Immobilized Affinity Ligand Techniques 80-83 (Academic Press,New York 1992). To 100 g beads, 100 ml of 0.1 M MES, (pH 4.5) containing10 mM dicarboxymethyl-PEG and 500 mg of EDC were added and incubated at25° C. with shaking (150 rpm) for 2 h. The PEG-modified beads werewashed with PBS, pH 7.4, and dried at 25° C. Anti-OVA IgG, anti-BG IgGand anti-E. coli IgG were attached to PEG and succinylated beads usingthe one-step EDC reaction. To 100 g of beads, 1 mg of antibody in 150 mLof 0.1 M 0.1 M MES buffer (pH 4.5) was added. EDC, 500 mg, was added andincubated at 25° C. for 2 hours. After washing antibody-modified beads(Ab-beads) 5 times with 50 ml PBST, BSA, 3% in PBST, was added andincubated overnight to block nonspecific binding sites on the glasssurface.

[0146] The BCA protein assay was employed to determine the amount ofprotein immobilized on the glass beads according to M. Bonde et al.,Direct dye binding-a quantitative assay for solid-phase immobilizedprotein, 200 Anal. Biochem. 195-198 (1992), prior to blocking with BSA.Antibody-modified beads, 8 g, were incubated with 5 mL of BCA reagentfor 30 min at 37° C. The amount of immobilized antibody was determinedbased on BSA as the standard.

[0147] Detection of Captured OVA, BG Spores and E. coli 0157:H7

[0148] Capture of OVA, BG spores and E. coli 0157:H7 onto Ab-beads wasdetected using a surface ELISA method. To 8 g of Ab-beads, 10 mL ofappropriate antigen dilution (OVA, BG, or E. coli 0157:H7) was added andincubated at 25° C. for 1 h on a shaker (150 rpm). Beads were thenwashed five times with 50 mL PBST (pH 7.2). Specific antibody (40 μg/10mL PBST) was added to the washed beads and incubated at 25° C. for 1 hat 150 rpm. Beads were washed five times with 50 mL PBST (pH 7.2) beforeaddition of tertiary antibody, anti-IgG-HRP, 1 μg in PBST. Beads wereincubated at 25° C. for 1 h at 150 rpm followed by washing five timeswith 50 mL PBST (pH 7.2). The substrate for HRP (5 mL of tetramethylbenzidine) was added to the beads and incubated in the dark for 15 min.The liquid, 1 ml, was removed from the beads and the absorbance at 370nm was measured with a Cary-100-Bio Spectrophotometer (Varian Inst.,Sugarland, Tex.).

[0149] Results and Discussion

[0150] Immobilized Antibodies

[0151] The results determined by the BCA protein assay indicated thatapproximately the same amount of protein was immobilized onto both thesuccinylated and PEG-modified beads. Considering the surface area of the3 mm glass beads (7.5×10⁻⁴ m²/g), the theoretical maximum amount ofimmobilized antibody was 15 mg antibody/m². These results are consistentwith other investigators, P. J. Soltys & M. R. Etzel, EquilibriumAbsorption of LDL and gold Immunoconjugates to Affinity MembranesContaining PEG Spacers, 21 Biomaterials 37-48 (2000), for a monolayer ofimmobilized antibody.

[0152] Comparison of Relative Capture Efficiency

[0153] The calibration plots for capturing OVA, BG spores and E.coli0157:H7 cells are shown in FIGS. 17A, B, and C. Signal at 370 nmindicates the amount of tertiary antibody, anti-IgG-HRP, bound to thesurface. Succinylated Ab-beads captured OVA, BG spores and E. coli0157:H7, but the capture efficiency was less than the PEG Ab-beads. Theslope of the PEG Ab-beads are higher compared to the succinylatedAb-beads for each antigen tested. The influence of a PEG spacer is moredramatic in the capture of E. coli O157:H7.

[0154] The observed difference in capture efficiency of PEG versussuccinylated Ab-beads can be explained by the long arm PEG provideswhich distances the antibodies from the support surface. This allowsgreater accessibility of the antigens to the immobilized antibodies,reducing the amount of steric hindrance. Since the total amount ofantibodies immobilized onto succinylated and PEG beads was similar, theantibodies immobilized via a spacer may have been able to capture theantigen more effectively.

EXAMPLE 20

[0155] Surface Modification. The capture ability of antibodies attachedto different spacers was investigated (see Table 4). Dextran (MW 37,500;Sigma, St. Louis, Mo.), polyethylene glycol-dicarboxylmethyl (PEG, MW3,400; Shearwater Polymers, Inc., Huntsville, Ala.), or polythreonine(MW 12,100; Sigma, St. Louis, Mo.) were used as spacers.

[0156] Anti-BSA Ab were bound to 2.8 μm tosyl-activated polystyreneDynalbeads (1 mg). Polythreonine (MW 12,100; Sigma) was used as spacerand attached to the beads by the method of M. Blake & B. C. Weimer,Immunomagnetic detection of Bacillus stearothermophilus spores in foodand environmental samples, 63 Appl. Environ. Microbiol. 1643-1646(1997). A total of 100 μl (10⁸ total beads) modified polystyrene beadswere used for each sample. Anti-OVA Ab at a concentration of 10¹⁶molecules/m² were bound to 3 mm glass beads by the method as describedabove. PEG was used as spacer and attached using the EDC facilitatedreaction. G. T. Hermanson, supra. Anti-B. globigii spore Ab were boundto 3 mm glass and 7 mm ceramic beads. Polythreonine was used as thespacer for the ceramic beads, whereas PEG and dextran were used asspacers with glass beads. G. T. Hermanson, supra. The antibodyconcentration was 10¹⁶ molecules/m² for all anti-B. globigii sporebeads. Anti-E. coli O157:H7 (Kirkegaard & Perry Laboratory,Gaithersburg, Md.) was attached to 3 mm glass beads using PEG as thespacer (as described above) at a concentration of 10¹³ molecules/m².Hybridization slides (2.4 cm² surface area) were also modified with thesame concentration of anti-E. coli O157:H7 antibodies using PEG as thespacer.

[0157] Detection in Static Environment. Eight grams of Ab modified beadswere placed into a 50 ml centrifuge tube and 10 ml of sample was addedto the beads. Samples were incubated on a rocker for 1 h at 25° C. Thesamples were washed six times each with 50 ml PBST (pH 5.8). SecondaryAb was added (total of 10¹² molecules of anti-E. coli O157:H7, 10¹³molecules of anti-OVA, 10¹³ molecules of anti-BSA, and 10¹² ofanti-Bacillus globigii) in 10 ml PBST and beads were again incubated for1 h. Samples were washed six times with 50 ml PBST (pH 5.8) andincubated with 10 ml of anti-IgG conjugated to horseradish peroxidase(Pierce Chemical Company, Rockford, Ill.; IgG-HRP, 1 μg/10 ml PBST, pH5.8). After the last wash step, beads were added to 5 ml of 1-Step TurboTMB-ELISA substrate (Pierce) and incubated in the dark for 20 min beforea reading was taken at A₃₇₀ using a Cary 100-Bio UV/Visiblespectrophotometer (Varian, Sugar Land, Tex.). Water blanks were used tozero the instrument.

[0158] Detection using Flow. Flow used a fluidized bed of beads, 8 g forthe small unit and 250 g for the large unit, with Ab covalently bound.To generate flow, a vacuum pump was used. The reagents were evacuatedfrom the bead cartridge through the top of the reactor at a constantrate of 0.4 L/min (or 5″ of Hg). As soon as all the liquid passed overthe beads the next reagent was allowed to flow through the reactor. Thiscontinued until all the reagents flowed across the beads. Just beforeadding the substrate (TMB) to the bead cartridge, the vacuum was turnedoff and the TMB was pulled into the reactor with a syringe. Once the TMBsolution covered the beads, the cartridge was sealed and placed in thedark for 20 min. To measure the color development at A₃₇₀, 1 ml of thesubstrate was placed in a cuvette. Water blanks were used to zero thespectrophotometer.

[0159] Four liters of 0.25 M sodium phosphate buffer (pH 7.0), or riverwater were spiked with 10⁶ total Bacillus globigii spores. A stainlesssteel module was filled with 250 g modified anti-B. globigii sporeceramic beads. The B. globigii spore solution was recycled over the 7 mmmodified ceramic beads for 60 min at 1, 2, and 4 L/min flow rates. Fivebeads were taken out every 15 min, replaced by 5 non-modified ceramicbeads, and capture ability of the beads investigated using the staticmethod. At the same time spore counts were determined on plate countagar.

[0160] The ability of the detection system to recover B. globigii sporesfrom various environmental and industrial water samples was alsoinvestigated. Samples were collected from various environmental andindustrial locations in Cache Valley, Utah: (A) Logan River water (pH8.4); (B) Gossner's Cheese Plant tank water (pH 9.2); (C) PBST (pH 7.2);and (D) Utah State University Dairy Plant slush tank (pH 7.2). Sampleswere tested in flow using 8 g of Ab modified beads. Standard curves weregenerated in these samples with pure cultures in buffer. The ability ofthe detection system to recover E. coli O157:H7 from meat extract andPBST samples was also investigated with 10⁴ total cells and anti-E. coliO157:H7 Ab attached to 3 mm glass beads via PEG. TABLE 4 List ofantibodies and their modifications used to capture bovine serum albumin(BSA), egg albumin (OVA), B. globigii spores, and E. coli O157:H7.Antibody Bead Size Spacer Matrices tested Anti-BSA polystyrene 2.8 μm  polythreonine PBS Anti-OVA Glass 3 mm PEG PBS Anti- Glass 3 mm PEG andEnvironmental B. globigii dextran and industrial spores Ceramic 7 mmpolythreonine water samples, 0.25 M sodium phosphate buffer, pH 7.2Anti-E. coli Glass 3 mm PEG PBS, meat O157:H7

[0161] Results

[0162] Static capture ability of modified beads. FIG. 18 shows thestandard curve obtained with anti-BSA-modified immunomagnetic beads andstatic detection. Ab modified polystyrene beads, 10⁸ total beads,successfully captured BSA. Very small amounts (<1 ng) of BSA can bedetected with these beads. The linear response of signal to BSA increasewas 99.7%, which makes this test very sensitive.

[0163]FIG. 19 shows the standard curve obtained for 3 mmPEG-anti-OVA-modified beads tested in static. The lower limit ofdetection is 0.2 μg. There is a linear response of signal increase toOVA increase between 0.2 to 4.0 μg. We did not test beyond 4 μg, becauseour objective was to develop a test that was sensitive on the lower end.

[0164] Flow Capture Ability of Modified Beads.

[0165]

[0166]FIG. 20A shows the standard curves obtained for B. globigii sporecapture at a constant sample flow of 0.4 m L/min using 8 g of 3 mm PEGmodified glass beads. A linear increase in signal was observed as sporeconcentration increased. FIG. 20B shows the Immuno capture of E. coliO157:H7 at a constant sample flow rate of 0.4 L/min with 8 g of 3 mm PEGmodified glass beads. To observe the influence of sample composition,PBST and sterile meat extract were used. Meat extract spiked with cellsconsistently showed a higher signal as compared to cells spiked intobuffer.

[0167]FIG. 21 shows the standard curve obtained for B. globigii sporesspiked into the various environmental and industrial water samples. Allsamples were sterilized prior to the addition of B. globigii spores. Nolinear response to increased spore concentration was observed usingriver or slush tank water. This results in a constant flat line as seenin FIG. 21. A linear response was observed using tank water fromGossner's Cheese Plant and PBST. The lower limit of detection for bothtank water and PBST was 1 spore/sample. However, the upper limit withPBST was 10⁵ total spores and for tank water 10³ total spores. Allenvironmental and industrial samples had pHs values ranging from 7.2 to9.2. The beads were active and captured spores over this range.

The subject matter claimed is:
 1. A method for capturing andconcentrating a selected antigen from an aqueous medium containing amixture of antigens comprising: (a) causing a first volume of theaqueous medium containing the mixture of antigens to flow through amodule containing at least two antibody-bead conjugates, wherein each ofthe antibody-bead conjugates comprises a bead, a polymeric spacercovalently coupled to the bead, and an antibody covalently coupled tothe polymeric spacer, wherein the antibody is configured for binding theselected antigen, at a first flow rate such that the antibody-beadconjugates form a fluidized bed and the selected antigen binds to theantibody-bead conjugates; (b) washing the antibody-bead conjugateshaving the selected antigen bound thereto by causing a wash medium toflow through the module at a second flow rate such that theantibody-bead conjugates having the selected antigen bound thereto forma fluidized bed; and (c) holding the washed antibody-bead conjugateshaving the selected antigen bound thereto in a second volume of a secondwash medium, wherein the second volume is smaller than the first volume.2. The method of claim 1 wherein the aqueous medium containing themixture of antigens comprises a food.
 3. The method of claim 1 whereinthe aqueous medium containing the mixture of antigens comprises anenvironmental sample.
 4. The method of claim 1 wherein the bead is aglass bead.
 5. The method of claim 1 wherein the bead is a ceramic bead.6. The method of claim 1 wherein the bead has a diameter of about 1 to 7millimeters.
 7. The method of claim 1 wherein the polymeric spacercomprises dextran.
 8. The method of claim 1 wherein the polymeric spacercomprises polyethylene glycol.
 9. The method of claim 1 wherein thepolymeric spacer comprises a polyamino acid.
 10. The method of claim 9wherein the polyamino acid comprises polythreonine.
 11. The method ofclaim 9 wherein the polyamino acid comprises polyserine.
 12. The methodof claim 1 wherein the antibody comprises a monoclonal antibody.
 13. Themethod of claim 1 wherein the antibody comprises a polyclonal antibody.14. The method of claim 1 wherein the antibody comprises an antibodyfragment.
 15. The method of claim 1 wherein flow is caused by pumping.16. The method of claim 1 wherein flow is caused by applying partialvacuum.
 17. The method of claim 1 wherein said first flow rate is about0.2 to 1.2 liters/minute.
 18. The method of claim 17 wherein the firstflow rate is about 0.3 to 0.7 liters per minute.
 19. A method forcapturing and concentrating a selected antigen from an aqueous mediumcontaining a mixture of antigens comprising: (a) causing a first volumeof the aqueous medium containing the mixture of antigens to flow througha module containing at least two antibody-bead conjugates, wherein eachof the antibody-bead conjugates comprises a 1-7 millimeter glass orceramic bead, a dextran or polyethylene glycol spacer covalently coupledto the bead, and an antibody covalently coupled to the spacer, whereinthe antibody is configured for binding the selected antigen, at a firstflow rate such that the antibody-bead conjugates form a fluidized bedand the selected antigen binds to the antibody-bead conjugates; (b)washing the antibody-bead conjugates having the selected antigen boundthereto by causing a wash medium to flow through the module at a secondflow rate such that the antibody-bead conjugates having the selectedantigen bound thereto form a fluidized bed; and (c) holding the washedantibody-bead conjugates having the selected antigen bound thereto in asecond volume of a second wash medium, wherein the second volume issmaller than the first volume.
 20. A method for detecting a selectedantigen in aqueous medium containing a mixture of antigens comprising:(a) causing the aqueous medium containing the mixture of antigens toflow through a module containing at least two antibody-bead conjugates,wherein each of the antibody-bead conjugates comprises a bead, apolymeric spacer covalently coupled to the bead, and an antibodycovalently coupled to the polymeric spacer, wherein the antibody isconfigured for binding the selected antigen, at a first flow rate suchthat the antibody-bead conjugates form a fluidized bed and the selectedantigen binds to the antibody-bead conjugates; (b) washing theantibody-bead conjugates having the selected antigen bound thereto bycausing a first wash medium to flow through the module at a second flowrate such that the antibody-bead conjugates having the selected antigenbound thereto form a fluidized bed; and (c) detecting the selectedantigen bound to the antibody-bead conjugates by enzyme-linkedimmunosorbent assay.
 21. The method of claim 20 wherein the aqueousmedium containing the mixture of antigens comprises a food.
 22. Themethod of claim 20 wherein the aqueous medium containing the mixture ofantigens comprises an environmental sample.
 23. The method of claim 20wherein the bead is a glass bead.
 24. The method of claim 20 wherein thebead is a ceramic bead.
 25. The method of claim 20 wherein the bead hasa diameter of about 1 to 7 millimeters.
 26. The method of claim 20wherein the polymeric spacer comprises dextran.
 27. The method of claim20 wherein the polymeric spacer comprises polyethylene glycol.
 28. Themethod of claim 20 wherein the polymeric spacer comprises a polyaminoacid.
 29. The method of claim 28 wherein the polyamino acid comprisespolythreonine.
 30. The method of claim 28 wherein the polyamino acidcomprises polyserine.
 31. The method of claim 20 wherein the antibodycomprises a monoclonal antibody.
 32. The method of claim 20 wherein theantibody comprises a polyclonal antibody.
 33. The method of claim 20wherein the antibody comprises an antibody fragment.
 34. The method ofclaim 20 wherein flow is caused by pumping.
 35. The method of claim 20wherein flow is caused by applying partial vacuum.
 36. The method ofclaim 20 wherein said first flow rate is about 0.2 to 1.2 liters/minute.37. The method of claim 36 wherein the first flow rate is about 0.3 to0.7 liters per minute.
 38. The method of claim 20 wherein said detectingthe selected antigen bound to the antibody-bead conjugates byenzyme-linked immunosorbent assay comprises: causing a medium comprisinga secondary antibody configured for binding the selected antigen to flowthrough the module such that the secondary antibody binds to theselected antigen bound to the antibody-bead conjugates; causing a secondwash medium to flow through the module such that secondary antibody thatdid not bind to the selected antigen is washed out of the module;causing a medium comprising a tertiary antibody-enzyme conjugateconfigured for binding the secondary antibody to flow through the modulesuch that the tertiary antibody-enzyme conjugate binds to the secondaryantibody bound to the selected antigen; causing a third wash medium toflow through the module such that tertiary antibody-enzyme conjugatethat did not bind to the secondary antibody is washed out of the module;causing a medium comprising an enzyme substrate to flow into the module,wherein the enzyme substrate is selected for being a substrate for thetertiary antibody-enzyme conjugate and being converted into a detectableproduct, and incubating the enzyme substrate in the module such that thedetectable product is produced; and measuring the detectable product.39. The method of claim 38 wherein the detectable product isluminescent.
 40. The method of claim 38 wherein the detectable productis fluorescent.
 41. The method of claim 38 wherein the measuring thedetectable product is carried out with a photomultiplier tube.
 42. Anapparatus for use in capturing and detecting antigens comprising: (a) ahousing comprising a wall defining an interior chamber and comprising aninlet opening for conducting a liquid medium into the interior chamberand an outlet opening for conducting the liquid medium out of theinterior chamber, wherein at least a portion of the wall is opticallytransparent; (b) at least two antibody-bead conjugates disposed in thehousing, each comprising a bead, a polymeric spacer covalently coupledto the bead, and an antibody coupled to the polymeric spacer; (c) aliquid circulation circuit coupled to the housing for conducting theliquid medium into the interior chamber through the inlet opening andfor conducting the liquid medium out of the interior chamber through theoutlet opening at a selected flow rate; and (d) a photomultiplier tubemounted adjacent to the optically transparent portion of the wall formeasuring photons produced in the interior chamber.
 43. The apparatus ofclaim 42 wherein the bead is a glass bead.
 44. The apparatus of claim 42wherein the bead is a ceramic bead.
 45. The apparatus of claim 42wherein the bead has a diameter of about 1 to 7 millimeters.
 46. Theapparatus of claim 42 wherein the polymeric spacer comprises dextran.47. The apparatus of claim 42 wherein the polymeric spacer comprisespolyethylene glycol.
 48. The apparatus of claim 42 wherein the polymericspacer comprises a polyamino acid.
 49. The apparatus of claim 48 whereinthe polyamino acid comprises polythreonine.
 50. The apparatus of claim48 wherein the polyamino acid comprises polyserine.
 51. The apparatus ofclaim 42 wherein the antibody comprises a monoclonal antibody.
 52. Theapparatus of claim 42 wherein the antibody comprises a polyclonalantibody.
 53. The apparatus of claim 42 wherein the antibody comprisesan antibody fragment.
 54. The apparatus of claim 42 wherein the liquidcirculation circuit comprises a pump.